In general, the invention features methods and compounds for increasing cell death.
Cell growth is a tightly regulated process. When the body has no need for new cells, but cells nonetheless divide in an unregulated manner, the result is cancer. Cancer therapies are directed at controlling the rapid proliferation of cells and/or controlling the differentiation rate of cells, as an undifferentiated cell is highly proliferative. One way in which the proliferation of cancer cells may be controlled is by killing such unregulated dividing cells.
The family of Bcl-2 proteins plays a central role in the regulation of cell life and death, acting by modulating apoptosis, a specific type of cell death. Some members of this family, for example, Bax, Bad, and Bak promote apoptosis, while other members of the family, for example, Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 inhibit apoptosis. The precise mechanism by which the various Bcl-2 family members promote either cell viability or cell death has not yet been resolved.
One method for treating cancer involves controlling the expression and/or activity of Bcl-2 family member proteins. In particular, methods that decrease the expression or activity of anti-apoptotic Bcl-2 family members or increase the expression or activity of pro-apoptotic Bcl-2 family members would be useful for treating cancer.
The present invention features methods and compounds for disrupting an interaction between a polypeptide containing a Bcl-2-homology-3 domain and another polypeptide, and for increasing cell death. The compounds of the present invention may be used as therapeutics to increase cell death in a desired cell, such as a cancer cell. These compounds are characterized by their ability to inhibit heterodimerization between pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins. Identified compounds may be especially useful in treating cancers that overexpress Bcl-2 protein family members.
Accordingly, in a first aspect, the invention features a chemical compound in a pharmaceutically acceptable carrier having the formula: 
where each of R1, R2, R4, and R5 is independently selected from the group consisting of hydrogen, alkoxyl, hydroxyl, and halogens; R3 is selected from the group consisting of N(CH3)2, phenyl, hydroxyl, alkoxyl, and halogens; R6 is selected from the group consisting of CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, and CH3; R7 is either hydrogen or an alkyl group; and the bond (a) is either a single or double bond.
In a preferred embodiment of the above aspect of the invention, the heterocyclic ring of the compound is substituted with a benzyl ring.
In another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is bromine; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In yet another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is chlorine; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In yet another preferred embodiment, in the compound, each of R1, R2, R3, R4, and R5 are hydrogen; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In still another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is N(CH3)2 ; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In other embodiments, the alkoxyl group of R1, R2, R4, R5, or R3 contains 10 or fewer carbons. Preferably the alkoxyl group of R1, R2, R4, R5, or R3 contains 4 or fewer carbons. Most preferably the alkoxyl group of R1, R2, R4, R5, or R3 is a methoxyl group.
In yet other embodiments of the above aspect of the invention, if R1 is hydrogen, then R2, R4, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R2 is hydrogen, then R1, R4, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R4 is hydrogen, then R1, R2, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R5 is hydrogen, then R1, R2, or R4 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R3 is bromine or chlorine, then R1, R2, R4, or R5 is not hydrogen; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R6 is CH(CH3)2, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R7 is not hydrogen; or the bond (a) is not a double bond. If R7 is hydrogen, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R6 is not CH(CH3)2; or the bond (a) is not a double bond. If the bond (a) is a double bond, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R6 is not CH(CH3)2; or R7 is not hydrogen.
In a second aspect, the invention features a chemical compound in a pharmaceutically acceptable carrier having the formula: 
where each of R1, R2, R4, and R5 is, independently, hydrogen, a halogen, or a phenyl group; and R3 is hydrogen or an alkyl group.
In a preferred embodiment of the second aspect of the invention, in the compound, each of R1, R4, and R5 is chlorine; R2 is bromine; and R3 is hydrogen.
In another preferred embodiment of the second aspect of the invention, in the compound, each of R1, R4, and R5 is chlorine; R2 is iodine; and R3 is hydrogen.
In yet another preferred embodiment of the second aspect of the invention, in the compound, R1 and R2 are iodine, R4, and R5 are chlorine; and R3 is hydrogen.
In other embodiments of the second aspect of the invention, if R1 is chlorine, then R4 or R5 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R4 is chlorine, then R1 or R5 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R5 is chlorine, then R1 or R4 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R2 is bromine, then R1, R4, or R5 is not chlorine; or R3 is not hydrogen. If R3 is hydrogen, then R1, R4, or R5 is not chlorine; or R2 is not bromine.
In a third aspect, the invention features a method for increasing cell death, involving the steps of:
(a) providing a cell predicted to be resistant to cell death, or to be at risk for resisting cell death; and
(b) contacting the cell with a chemical compound having the formula: 
where each of R1, R2, R4, and R5 is independently selected from the group consisting of hydrogen, alkoxyl, hydroxyl, and halogens; R3 is selected from the group consisting of N(CH3)2, phenyl, hydroxyl, methoxyl, and halogens; R6 is selected from the group consisting of CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, and CH3; R7 is either hydrogen or an alkyl group; and the bond (a) is either a single or double bond.
In a preferred embodiment of the above aspect of the invention, in the compound, the heterocyclic ring of the compound is substituted with a benzyl ring.
In another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is bromine; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In yet another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is chlorine; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In yet another preferred embodiment, in the compound, each of R1, R2, R3, R4, and R5 are hydrogen; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In still another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is N(CH3)2; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In other embodiments, in the compound,the alkoxyl group of R1, R2, R4, R5, or R3 contains 10 or fewer carbons. Preferably the alkoxyl group of R1, R2, R4, R5, or R3 contains 4 or fewer carbons. Most preferably the alkoxyl group of R1, R2, R4, R5, or R3 is a methoxyl group.
In yet other embodiments of the above aspect of the invention, in the compound, if R1 is hydrogen, then R2, R4, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R2 is hydrogen, then R1, R4, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R4 is hydrogen, then R1, R2, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R5 is hydrogen, then R1, R2, or R4 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R3 is bromine or chlorine, then R1, R2, R4, or R5 is not hydrogen; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R6 is CH(CH3)2, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R7 is not hydrogen; or the bond (a) is not a double bond. If R7 is hydrogen, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R6 is not CH(CH3)2; or the bond (a) is not a double bond. If the bond (a) is a double bond, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R6 is not CH(CH3)2; or R7 is not hydrogen.
In a fourth aspect, the invention features a method for increasing cell death, said method involving the steps of:
(a) providing a cell predicted to be resistant to cell death, or to be at risk for resisting cell death; and
(b) contacting the cell with a chemical compound having the formula: 
where each of R1, R2, R4, and R5 is, independently, hydrogen, a halogen, or a phenyl group; and R3 is hydrogen or an alkyl group.
In a preferred embodiment of the fourth aspect of the invention, in the compound, each of R1, R4, and R5 is chlorine; R2 is bromine; and R3 is hydrogen.
In another preferred embodiment of the fourth aspect of the invention, in the compound, each of R1, R4, and R5 is chlorine; R2 is iodine; and R3 is hydrogen.
In yet another preferred embodiment of the fourth aspect of the invention, in the compound, R1 and R2 are iodine, R4, and R5 are chlorine; and R3 is hydrogen.
In other embodiments of the fourth aspect of the invention, in the compound, if R1 is chlorine, then R4 or R5 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R4 is chlorine, then R1 or R5 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R5 is chlorine, then R1 or R4 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R2 is bromine, then R1, R4, or R5 is not chlorine; or R3 is not hydrogen. If R3 is hydrogen, then R1, R4, or R5 is not chlorine; or R2 is not bromine.
In one embodiment of the third or fourth aspect of the invention, the cell expresses a pro-apoptotic and/or anti-apoptotic protein. Preferably the pro-apoptotic protein is selected from the group consisting of pro-apoptotic proteins containing a Bcl-2-homology-domain-3, such as Bax, Bak, Bok, Bad, Bid, Bik, Bim, or Hrk. In another embodiment, the anti-apoptotic protein is chosen from the group consisting of Bcl-2, Bcl-xL, Mcl-1, and Bcl-w. In another embodiment, the compound is substantially pure. In another embodiment, the compound is in a pharmaceutically acceptable carrier.
In another embodiment of the third and fourth aspects of the invention, the cell is mammalian. Preferably the cell is a rodent cell, such as a mouse or rat cell. Most preferably, the cell is a human cell. In another embodiment, the cell is a cancer cell.
In a fifth aspect, the invention features a method for treating a condition in a subject, involving administering a chemical compound having the formula: 
where each of R1, R2, R4, and R5 is independently selected from the group consisting of hydrogen, alkoxyl, hydroxyl, and halogens; R3 is selected from the group consisting of N(CH3)2, phenyl, alkoxyl, hydroxyl, and halogens; R6 is selected from the group consisting of CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, and CH3; R7 is either hydrogen or an alkyl group; and the bond (a) is either a single or double bond.
In a preferred embodiment of the above aspect of the invention, in the compound, the heterocyclic ring of the compound is substituted with a benzyl ring.
In another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is bromine; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In yet another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is chlorine; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In yet another preferred embodiment, in the compound, each of R1, R2, R3, R4, and R5 are hydrogen; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In still another preferred embodiment, in the compound, each of R1, R2, R4, and R5 are hydrogen; R3 is N(CH3)2 ; R6 is CH(CH3)2; R7 is hydrogen; and the bond (a) is a double bond.
In other embodiments, in the compound, the alkoxyl group of R1, R2, R4, R5, or R3 contains 10 or fewer carbons. Preferably the alkoxyl group of R1, R2, R4, R5, or R3 contains 4 or fewer carbons. Most preferably the alkoxyl group of R1, R2, R4, R5, or R3 is a methoxyl group.
In yet other embodiments of the above aspect of the invention, in the compound, if R1 is hydrogen, then R2, R4, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R2 is hydrogen, then R1, R4, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R4 is hydrogen, then R1, R2, or R5 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R5 is hydrogen, then R1, R2, or R4 is not hydrogen; or R3 is not bromine or chlorine; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R3 is bromine or chlorine, then R1, R2, R4, or R5 is not hydrogen; or R6 is not CH(CH3)2; or R7 is not hydrogen; or the bond (a) is not a double bond. If R6 is CH(CH3)2, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R7 is not hydrogen; or the bond (a) is not a double bond. If R7 is hydrogen, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R6 is not CH(CH3)2; or the bond (a) is not a double bond. If the bond (a) is a double bond, then R1, R2, R4, or R5 is not hydrogen; or R3 is not chlorine or bromine; or R6 is not CH(CH3)2; or R7 is not hydrogen.
In a sixth aspect, the invention features a method for treating a condition in a subject, involving administering a chemical compound having the formula: 
where each of R1, R2, R4, and R5 is, independently, hydrogen, a halogen, or a phenyl group; and R3 is hydrogen or an alkyl group.
In a preferred embodiment of the sixth aspect of the invention, in the compound, each of R1, R4, and R5 is chlorine; R2 is bromine; and R3 is hydrogen.
In another preferred embodiment of the sixth aspect of the invention, in the compound, each of R1, R4, and R5 is chlorine; R2 is iodine; and R3 is hydrogen.
In yet another preferred embodiment of the sixth aspect of the invention, in the compound, R1 and R2 are iodine, R4, and R5 are chlorine; and R3 is hydrogen.
In other embodiments of the sixth aspect of the invention, in the compound, if R1 is chlorine, then R4 or R5 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R4 is chlorine, then R1 or R5 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R5 is chlorine, then R1 or R4 is not chlorine; or R2 is not bromine; or R3 is not hydrogen. If R2 is bromine, then R1, R4, or R5 is not chlorine; or R3 is not hydrogen. If R3 is hydrogen, then R1, R4, or R5 is not chlorine; or R2 is not bromine.
In another embodiment of the fifth or sixth aspect of the invention, the condition is any condition in which the occurrence of cell death is too low. Preferably the condition is cancer, such as prostate cancer, breast cancer, gastrointestinal cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, stomach cancer, or a brain tumor, or a leukemia, lymphoma, or carcinoma.
In another embodiment, the subject is a mammal. Preferably the subject is a rodent, such as a mouse or rat. Most preferably the subject is a human.
In another embodiment of the fifth or sixth aspect of the invention, at least two of the compounds are administered, preferably, simultaneously.
In a seventh aspect, the invention features a method for identifying a compound that disrupts an interaction between a first polypeptide containing a Bcl-2-homology-3 domain and a second polypeptide, involving the steps of: providing a test compound, a first polypeptide containing a Bcl-2-homology-3 domain, and a second polypeptide; combining the test compound, first polypeptide, and second polypeptide; and measuring the interaction between the first and second polypeptides, relative to a control that comprises only the first and second polypeptides, where the measuring is done using a fluorescence polarization assay, and where a decrease in the interaction between the first and second polypeptides identifies the test compound as disrupting an interaction between the first and second polypeptides.
In an eighth aspect, the invention features a method for identifying a compound that increases cell death, involving the steps of: contacting a cell with a test compound that disrupts the interaction between a first polypeptide containing a Bcl-2-homology-3 domain and a second polypeptide; and measuring cell death relative to a control cell, where the measuring is done using a fluorescence polarization assay, and where an increase in cell death indicates that the test compound increases cell death.
In one embodiment of the seventh or eighth aspect of the invention, the first polypeptide is chosen from the group consisting of Bax, Bak, Bok, Bad, Bid, Bik, Bim, and Hrk.
By xe2x80x9cincreasing cell deathxe2x80x9d is meant increasing the number of cells that undergo cell death relative to a control cell that is not contacted with any test compounds. Preferably cell death is increased 10% relative to a control. More preferably cell death is increased 50% relative to a control. Most preferably cell death is increased is increased 90% relative to a control.
Cell death may be increased by contacting a cell with a test compound. An increase in cell death may be identified by determining the ATP level in a cell that has been contacted with a test compound, such as a small molecule from a chemical library, and comparing it to the ATP level in a control cell, for example, according to the methods of Crouch et al. (J. Immunol. Methods 160:81-8, 1993) Storer et al. (Mutat. Res. 368:59-101, 1996) or Cree et al. (Toxicol. In Vitro 11:553-556, 1997). Cell death is increased when the ATP level of a cell contacted with a test compound decreases more than the ATP level of a control cell. Cell death may also be measured using any of the assays described herein.
By xe2x80x9cthe occurrence of cell death is too lowxe2x80x9d or xe2x80x9cresistant to cell deathxe2x80x9d is meant that a cell or a population of cells does not undergo cell death under appropriate conditions. For example, normally a cell will die upon exposure to cytotoxic agents, such as chemotherapeutic agents or ionizing radiation. However, when the occurrence of cell death is too low, for example, in a subject having cancer, the cell or a population of cells may not undergo cell death in response to contact with cytotoxic agents. In addition, the occurrence of cell death may be too low when the number of proliferating cells exceeds the number of cells undergoing cell death, as occurs in cancer when such cells do not properly differentiate.
By xe2x80x9ccell deathxe2x80x9d is meant the death of a cell by either apoptosis or necrosis. Cell death may be characterized by cellular ATP depletion. Preferably the cell is depleted of ATP 10% relative to a control cell. More preferably the cell is depleted of ATP 50% relative to a control cell. Most preferably the cell is depleted of ATP 90% relative to a control cell. The level of cell death may be measured by determining the amount of ATP in a cell.
By xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate the level of cell death by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally-occurring organic molecules, nucleic acid molecules, and components thereof. Test compounds also include salts of any of the above chemicals.
By xe2x80x9capoptosisxe2x80x9d is meant cell death characterized by any of the following properties: nuclear condensation, DNA fragmentation, membrane blebbing, or cell shrinkage.
By an xe2x80x9canti-apoptotic-proteinxe2x80x9d is meant a protein which when expressed in a cell decreases cell death, as compared to a cell that does not express the anti-apoptotic protein. Preferably cell death in the cell containing the anti-apoptotic protein is decreased 10% relative to a control. More preferably cell death in the cell containing the anti-apoptotic protein is decreased 50% relative to a control. Most preferably cell death in the cell containing the anti-apoptotic protein is decreased 90% relative to a control.
By a xe2x80x9cpro-apoptotic proteinxe2x80x9d is meant a protein that when expressed in a cell increases cell death, as compared to a cell that does not express the pro-apoptotic protein. Preferably cell death in the cell containing the pro-apoptotic protein is increased 10% relative to a control. More preferably cell death in the cell containing the pro-apoptotic protein is increased 50% relative to a control. Most preferably cell death in the cell containing the pro-apoptotic protein is increased 90% relative to a control.
By xe2x80x9cinteractsxe2x80x9d is meant a compound that recognizes and binds to an anti-apoptotic protein but which does not substantially recognize and bind to other molecules in a sample.
By xe2x80x9cdisrupts an interactionxe2x80x9d is meant that a test compound decreases the ability of two polypeptides to interact with each other. Preferably the disruption results in a 50% decrease in the ability of the polypeptides to interact with each other. More preferably disruption results in a 75% decrease in the ability of the polypeptides to interact with each other. Most preferably the disruption results in a 99% decrease in the ability of the polypeptides to interact with each other.
As used herein, by xe2x80x9csubstantially purexe2x80x9d is meant a compound that is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, said compound, e.g., a compound from a chemical library. A purified compound may be obtained using methods known to those in the fields of medicinal and organic chemistry.
By xe2x80x9ccontaining a Bcl-2-homology-3 domainxe2x80x9d or xe2x80x9ccontaining a BH3 domainxe2x80x9d or a xe2x80x9cBH3 peptidexe2x80x9d is meant a polypeptide that is substantially identical to the amino acid sequence LRRIGDEF (SEQ ID NO: 1).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide exhibiting at least 60%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid sequence.
By xe2x80x9ctreatingxe2x80x9d is meant to submit or subject an animal, cell, lysate or extract derived from a cell, or a molecule derived from a cell to a compound that increases cell death.
By xe2x80x9cconditionxe2x80x9d is meant a state of being or feeling. Conditions include, but are not limited to, cancer, for example, prostate cancer, breast cancer, gastrointestinal cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, stomach cancer, or a brain tumor, or a leukemia, lymphoma, or carcinoma, or the symptoms associated with cancer.
By a xe2x80x9cdosage sufficient to increase cell deathxe2x80x9d is meant an amount of a chemical compound or small molecule which when administered to a subject will increase cell death. Preferably cell death is increased in the subject 10% relative to an untreated subject. More preferably cell death is increased in the subject 50% relative to an untreated subject. Most preferably cell death is increased in the subject 90% relative to an untreated subject.
By a xe2x80x9cderivativexe2x80x9d is meant a structural derivative having a chemical modification of the compound which does not increase the ultimate level of cell death, but which does enhance bioavailability, solubility, or stability in vivo or ex vivo or which reduces the toxicity or dosage required. Such modifications are known to those skilled in the field of medicinal chemistry.
As used herein, by xe2x80x9cmeasuring cell deathxe2x80x9d is meant determining if a cell is dying in the presence of a compound compared to a cell that is not in the presence of the compound (control cell). Cell death can be measured by determining cellular ATP levels, wherein a cell that is undergoing cell death has a decreased level of cellular ATP compared to a control cell. Cell death may also be measured by staining with a vital dye, for example, trypan blue, wherein a cell that is dead will be stained with the vital dye, and a cell that is not dead will not be stained with the dye. Cell death can also be measured by contacting a cell with Hoescht stain and viewing it for morphological indications of cell death. Such indications include nuclear fragmentation. Other assays for measuring cell death are described herein.
By xe2x80x9cfluorescence polarization assayxe2x80x9d is meant an assay in which an interaction between two polypeptides is measured. In this assay, one polypeptide is labeled with a fluorescent tag, and this polypeptide emits nonpolarized light when excited with polarized light. Upon an interaction of the tagged polypeptide with another polypeptide, the polarization of emitted light is increased, and this increased polarization of light can be detected.
The present invention provides a number of advantages. For example, the methods described herein allow for an increase in cell death or a disruption of the interaction between Bcl-2 family members. The invention also provides compounds and methods for treating diseases in which a cell is resistant to cell death. These compounds and methods can be used to treat conditions such as cancer.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.